Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143Parker Institute for Cancer Immunotherapy, San Francisco, CA 94143Life Science Center, Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan

NK cells in NKp46-CreERT2 Tg mice carrying the Rosa26-tdTomato allele or the Rosa26-YFP alleles express tdTomato or YFP after tamoxifen administration. (A) Schematic representation of the strategy used to generate NKp46-CreERT2 Tg mice. An IRES-CreERT2 cassette was inserted into the 3′ untranslated region of exon 7 of the mouse Ncr1 gene on mouse chromosome 7 in a C57BL/6 BAC. (B–D) NKp46-CreERT2 Tg mice with a heterozygous Rosa26-tdTomato allele were treated with tamoxifen for 5 consecutive days and immune cells were analyzed after the last tamoxifen administration. (B) Expression of tdTomato in NK cells (DX5+NKp46+TCRβ−B220−); T cells (NKp46−TCRβ+B220−); B cells (NKp46−TCRβ−B220+); and non-NK, non-T, and non-B cells (NKp46−TCRβ−B220−, predominantly myeloid cells) in the spleen. (C) Expression of tdTomato in NK precursor cells (TCRβ−CD19−CD11b−DX5−NKp46−CD122+) and immature (CD11b−CD27+), intermediate (CD11b+CD27+), and mature (CD11b+CD27+) NK cells (TCRβ−CD19−DX5+NKp46+) in the bone marrow. (D) Expression of tdTomato in NK cells (TCRβ−CD19−DX5+NKp46+CD122+), liver-resident NK cells (TCRβ−CD19−DX5−NKp46+CD122+), ILC1 (TCRβ−CD19−DX5−NKp46+CD11b−CD27+CD122low), and NKp46+ ILC3 (TCRβ−CD19−DX5−NKp46+CD11b−CD27−CD122low that are potentially ILC3) in the liver. Data are representative of two to three experiments (n = 2 in each experiment). (E and F) NKp46-CreERT2 Tg mice with a heterozygous Rosa26-YFP allele (E and F) and homozygous Rosa26-YFP alleles (F) were treated with tamoxifen for 5 d, and then splenocytes were analyzed. (E) Expression of YFP in NK cells; T cells; B cells; and non-NK, non-T, and non-B cells (predominantly myeloid cells) in the spleen. (F) Expression of YFP by NK cells in the spleen. Data are representative of more than five experiments (n = 2–6 in each experiment). Bold and thin lines represent cells in mice treated with tamoxifen and cells in untreated mice, respectively.

NK cells differentiate into memory NK cells and cytokine-activated NK cells after MCMV infection. NKp46-CreERT2 Tg mice with a heterozygous Rosa26-YFP allele (A) or homozygous Rosa26-YFP alleles (B–H) were treated with tamoxifen on days 0–4 and infected or not with MCMV on day 0. (A) The number of YFP+ NK cells and YFP− NK cells in the blood over the course of infection. Data were pooled from two experiments (n = 7 in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.005 versus Ly49H− cells. (B) Expression of Ly49H and KLRG1 on YFP+ NK cells in the spleens of uninfected and infected mice on day 27 p.i. The percentages of Ly49H+KLRG1high NK cells and Ly49H−KLRG1high NK cells are shown. Expression of KLRG1 on YFP+ NK cells in the spleens of naive uninfected mice and on YFP+Ly49H+ memory NK cells and cytokine-activated YFP+Ly49H− NK cells from MCMV-infected mice. Bold solid lines, bold dashed lines, thin solid lines, and thin dashed lines represent memory NK cells, cytokine-activated NK cells, naive Ly49H+ NK cells, and naive Ly49H− NK cells, respectively. (C) Phenotype of YFP+ NK cells in the spleens of naive uninfected mice, and YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the spleens of infected mice on day 25–32 p.i. (D) Expression of Granzyme B in YFP+ NK cells in the spleens of naive uninfected mice, and in memory YFP+Ly49H+KLRG1high NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the spleens of infected mice on day 32 p.i. Bold solid lines, bold dashed lines, and thin lines represent memory NK cells, cytokine-activated NK cells, and naive NK cells, respectively. Filled histograms represent staining with an isotype-matched control Ig. Data are representative of more than five experiments (n = 2–6 in each experiment). (E) The percentages of YFP+ NK cells in naive uninfected mice on days 25–27 after tamoxifen treatment and the percentages of YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the organs of tamoxifen-treated infected mice on days 25–27 p.i. *, P < 0.05; **, P < 0.01. (F) The percentages of YFP+Ly49H+ NK cells and YFP+Ly49H− NK cells expressing a memory phenotype KLRG1highLy6C+DNAM-1low in the organs of tamoxifen-treated infected mice on days 25–27 p.i. Data are pooled from six experiments (n = 4–8 in each group). *, P < 0.01 versus memory cells. (G) Expression of CD49d on YFP+ NK cells in the blood of naive uninfected mice on day 25 after tamoxifen treatment and on YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the blood of infected mice on day 25 p.i. Bold solid lines, bold dashed lines, and thin lines represent memory NK cells, cytokine-activated NK cells, and naive NK cells, respectively. A filled histogram represents staining with a control isotype-matched control Ig. Mean fluorescence intensity (MFI) of CD49d staining of NK cells are shown in H. Data are representative of two experiments (n = 4–6 in each group). *, P < 0.01 versus memory cells. P-values were calculated by a Student’s t test. Error bars show SEM.

IL-12 is required for the optimal differentiation of both memory Ly49H+ NK cells and cytokine-activated Ly49H− NK cells during MCMV infection

The differentiation of memory Ly49H+ NK cells during MCMV infection requires IL-12 (Sun et al., 2012). We examined the requirement of IL-12 for the generation of cytokine-activated Ly49H−KLRG1high NK cells in vivo during MCMV infection. When NKp46-CreERT2 x Rosa26-YFP Tg mice were infected with MCMV and treated with tamoxifen, neutralization of IL-12 on the day before MCMV infection and on days 3 and 6 p.i. significantly impaired the differentiation of both YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells (Fig. 3). The neutralization of IL-12 did not affect the phenotype of Ly49H+KLRG1high memory NK cells or cytokine-activated Ly49H−KLRG1high NK cells (unpublished data), but did affect the magnitude of their response. These findings indicate that IL-12 is essential for the optimal differentiation of both Ly49H+ memory NK cells and cytokine-activated Ly49H− NK cells during MCMV infection.

IL-12 is required for the optimal differentiation of memory NK cells and cytokine-activated NK cells. NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated daily with tamoxifen on days 0–4 and infected with MCMV on day 0. Mice were injected with 200 µg control Ig or anti–IL-12 neutralizing mAb on the day before infection and on day 3 and 6 p.i. The percentages of YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the spleens on day 26 p.i. are shown. Data are pooled from two experiments (n = 4–5 in each group). *, P < 0.01; **, P < 0.005 versus control Ig. P-values were calculated by a Student’s t test. Error bars show SEM.

Cytokine-activated NK cells preferentially persist in an MCMV-free environment. (A) NKp46-CreERT2 Tg mice with a heterozygous Rosa26-tdTomato allele or homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with MCMV on day 0. On day 27 after treatment with tamoxifen, tdTomato+ NK cells from naive uninfected mice were mixed with YFP+ NK cells from naive uninfected mice and with MCMV-primed YFP+ NK cells from infected mice, and then adoptively transferred into Rag1-deficient B6 mice. Donor NK cells were analyzed on day 5 after the transfer. (B) Expression of Ly49H and KLRG1 on donor tdTomato+ NK cells and YFP+ NK cells before transfer and in the spleens of Rag1-deficient recipient mice on day 5 after transfer. Splenocytes on day 5 after transfer were fixed and permeabilized for staining of Ki67. Thus, the fluorescence of tdTomato and YFP decreases as compared with before transfer. Data are representative of two experiments (n = 3–4 in each experiment). (C) The number of donor NK cells in the spleens of Rag1-deficient recipient mice on day 5 after transfer. The y axis represents the number of donor NK cells detected in the spleens of Rag1-deficient mice on day 5 compared with the number of donor NK cells adoptively transferred into Rag1-defiicent mice. *, P < 0.01 versus memory YFP+ cells. (D) The percentages of Ki67+ donor NK cells on day 5. Data were pooled from two experiments (n = 6–7 in each group). *, P < 0.05 versus naive tdTomato+ cells and naive YFP+ cells. (E–G) NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with MCMV on day 0. On day 27 after tamoxifen treatment, YFP+ NK cells isolated from naive uninfected mice and MCMV-primed YFP+ NK cells were adoptively transferred into Rag1-deficient B6 mice. Donor NK cells were analyzed on day 5 after the transfer. (E) Expression of Bcl-2 in donor NK cells on day 5. Bold solid lines, bold dashed lines, and thin lines represent memory NK cells, cytokine-activated NK cells, and naive NK cells, respectively. A filled histogram represents staining with an isotype-matched control Ig. Data were representative of two experiments (n = 2–3 in each experiment). MFI of Bcl-2 staining in NK cells is shown in F. (G) The percentages of Annexin V+ donor NK cells on day 5. Data were pooled from two experiments (n = 5 in each group). *, P < 0.05 versus memory cells. (H–K) NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with MCMV on day 0. (H and I) On day 25 after tamoxifen treatment, YFP+ NK cells from naive uninfected mice or MCMV-primed YFP+ NK cells were mixed with CD45.1+ WT B6 NK cells from naive uninfected mice, labeled with CellTrace Violet, and cultured in the presence of 2 or 10 ng/ml IL-15 for 4 d. (H) Cell divisions of NK cells on day 4. Filled histograms represent naive YFP+ NK cells. Bold and thin lines represent YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells, respectively. The number of cell divisions of NK cells was quantified in I. Data are representative of two experiments (n = 3–6 in each experiment). *, P < 0.05 versus memory cells. (J) Expression of CD122 and CD132 on YFP+ NK cells in the spleens of naive uninfected mice and YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells in the spleens of infected mice on days 25–27 p.i. Bold solid lines, bold dashed lines, and thin lines represent memory NK cells, cytokine-activated NK cells, and naive NK cells, respectively. Filled histograms represent staining with an isotype-matched control Ig. Data are representative of more than three experiments (n = 2–3 in each experiment). MFI of CD122 and CD132 staining of NK cells are shown in K. Data were pooled from three experiments (n = 6–7 in each group). *, P < 0.005 versus naive cells. P-values were calculated by a Student’s t test. Error bars show SEM.

Memory NK cells exert augmented antitumor activity. NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or not with WT MCMV on day 0. (A and B) Degranulation (A) and IFN-γ production (B) of naive YFP+ NK cells from naive uninfected mice and YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells from infected mice after culture in the presence of IL-12 and IL-18, stimulation with anti-NK1.1 mAb, and co-culture with RMA transfectants expressing m157 or Rae1γ. Data are representative of three experiments (n = 3–4 in each experiment). *, P < 0.05. (C) Splenic YFP+ NK cells from naive uninfected mice, and YFP+Ly49H+KLRG1high memory NK cells and cytokine-activated YFP+Ly49H−KLRG1high NK cells from infected mice were isolated on day 26 p.i. 15,000 YFP+ NK cells were transferred into T cell–depleted DAP10 and DAP12-double deficient recipient B6 mice. CellTrace Violet–labeled RMA-Rae1γ transfectants and RMA-S cells were mixed with RMA cells at a 1:1:1 (105 of each cell line) ratio, and injected intraperitoneally into these recipient mice on the day of NK cell transfer. An aliquot of the mixed tumor cells was cultured as a control. Proportions of these tumor cells (RMA, H-2Kb+ CellTrace Violet−; RMA-Rae1γ transfectants, H-2Kb+ CellTrace Violet+; and RMA-S cells, H-2Kb− CellTrace Violet+) in the peritoneal cavity were analyzed at 48 h after the injection. Data are representative of two experiments (n = 3 in each experiment). (D) NKG2D-mediated cytotoxicity against RMA-Rae1γ transfectants was quantified as the number of RMA-Rae1γ transfectants compared with the number of RMA cells detected in the peritoneal cavity at 48 h after injection. The y axis represents the clearance of RMA-Rae1γ transfectants in the peritoneal cavity of recipient mice receiving donor NK cells normalized to that of recipient mice not receiving donor NK cells expressed as a relative quantity. Data were pooled from two experiments (n = 6 in each group). *, P < 0.01 versus memory cells. P-values were calculated by a Student’s t test. Error bars show SEM.

Ly49H signaling is required for the differentiation of functional memory NK cells. NKp46-CreERT2 Tg mice with homozygous Rosa26-YFP alleles were treated with tamoxifen for 5 d and infected or uninfected with WT or Δm157 MCMV on day 0. (A) Phenotype of YFP+ NK cells in the spleens of naive uninfected mice, YFP+Ly49H+KLRG1high memory NK cells in the spleens of WT MCMV-infected mice, and MCMV-primed YFP+Ly49H+KLRG1high NK cells in the spleens of Δm157 MCMV-infected mice on days 25–27 p.i. The percentages of Ly49H+KLRG1high NK cells are shown. (B and C) Degranulation (B) and IFN-γ production (C) of YFP+ Ly49H+ NK cells from naive uninfected mice, YFP+Ly49H+KLRG1high memory NK cells from WT MCMV-infected mice, and MCMV-primed YFP+Ly49H+KLRG1high NK cells from Δm157 MCMV-infected mice after culture with IL-12 and IL-18, stimulation with anti-NK1.1 mAb, and co-culture with RMA cells expressing m157. Data are representative of two experiments (n = 3 in each experiment). *, P < 0.05. P-values were calculated by a Student’s t test. Error bars show SEM.

Discussion

Here, by using novel transgenic mice carrying inducible Cre under the control of expression by the Ncr1 gene, we found that two distinct NK cell subsets are generated by MCMV infection. One is an activating receptor ligand–driven memory NK cell and the other is an inflammatory cytokine-activated NK cell. Further, we directly compared the in vitro and in vivo functional properties and fate of activating receptor signal–driven Ly49H+KLRG1high memory NK cells versus cytokine-activated Ly49H−KLRG1high NK cells, both of which were generated in the same environment. Although prior studies have addressed the phenotypes, effector functions, persistence, and tissue distribution of mouse NK cells generated after MCMV infection in vivo or by in vitro activation with cytokines and then adoptive transfer into congenic mice (Cooper et al., 2009; Sun et al., 2009, 2010; Ni et al., 2012; van Helden et al., 2012), no studies have directly compared the functions and tracked the fate of activating receptor signal–driven memory NK cells versus cytokine-activated NK cells that are generated in vivo in a physiologically relevant viral infection. Our studies revealed that activating receptor signal–driven Ly49H+KLRG1high memory NK cells have augmented effector functions in vitro and in vivo, whereas cytokine-activated Ly49H−KLRG1high NK cells persist longer in an MCMV-free environment.

MCMV establishes latency after clearance of the primary infection and replicates chronically in the salivary glands (Vliegen et al., 2003). Prior studies have demonstrated that NK cells, T cells, and neutralizing antibodies against MCMV are critical for preventing recurrence and dissemination of MCMV from the salivary glands of latently infected mice (Jonjić et al., 1994; Polić et al., 1998). One of the notable differences between Ly49H+KLRG1high memory NK cells and cytokine-activated Ly49H−KLRG1high NK cells is their tissue distribution. Although both Ly49H+ and Ly49H− NK cells exist in the salivary glands of naive uninfected mice, Ly49H+KLRG1high memory NK cells, but not cytokine-activated Ly49H−KLRG1high NK cells, predominantly resided in the salivary glands of MCMV-infected mice. These findings suggest that the preferential accumulation of Ly49H+KLRG1high memory NK cells in the salivary glands might contribute to suppression of reactivation of MCMV in latently infected hosts. Despite our findings that Ly49H+KLRG1high memory NK cells predominate over cytokine-activated Ly49H−KLRG1high NK cells in the organs of MCMV-infected mice, cytokine-activated Ly49H−KLRG1high NK cells exhibited better persistence than memory Ly49H+KLRG1high NK cells in MCMV-free recipient mice. A possible explanation is that persistence of MCMV-specific Ly49H+KLRG1high memory NK cells is dependent on the equilibrium between viral replication and dissemination of MCMV and subsequent reactivation, causing proliferation of Ly49H+KLRG1high memory NK cells.

The previously described mouse cytokine-induced memory-like NK cells were generated by culturing NK cells in vitro with pharmacological amounts of a combination of cytokines (Cooper et al., 2009; Ni et al., 2012). However, there is no evidence that these cytokine-induced memory-like NK cells are generated in vivo in physiological situations. Here, we demonstrated that cytokine-activated Ly49H−KLRG1high NK cells are generated in vivo after MCMV infection. However, the functional properties of in vivo MCMV-induced cytokine-activated NK cells are different than the in vitro–generated cytokine-induced memory-like NK cells. The in vitro–derived cytokine-induced memory-like NK cells displayed enhanced IFN-γ production in response to ex vivo stimulation with cytokines, cross-linking of activating receptors, and tumor target cells, although they did not exert enhanced cytotoxicity (Cooper et al., 2009; Ni et al., 2012). However, we have demonstrated that in vivo MCMV-induced cytokine-activated NK cells display both enhanced degranulation and IFN-γ production in response to stimulation by cross-linking activating receptors and tumor target cells, but demonstrated poor IFN-γ production in response to IL-12 plus IL-18. Thus, the in vivo–generated cytokine-activated NK cells are not necessarily the counterparts of the cytokine-induced memory-like NK cells that were generated by in vitro culture.

HCMV-seropositive healthy individuals homozygously carrying the KLRC2 gene that encodes NKG2C show a significantly higher frequency and number of CD94-NKG2C+ NK cells than those carrying a heterozygous deletion of KLRC2 (Noyola et al., 2012; Muntasell et al., 2013). In KLRC2 hemizygous individuals, KLRC2+/− NK cells exhibit diminished calcium influx, degranulation, and proliferation upon NKG2C ligation compared with KLRC2+/+ NK cells (Muntasell et al., 2013). In response to acute HCMV infection, NK cells in patients transplanted with umbilical cord blood obtained from donors carrying a homozygous deletion of KLRC2 demonstrated an expansion of mature NK cells expressing an activating KIR, suggesting that receptors other than CD94-NKG2C may also drive NK cell differentiation (Della Chiesa et al., 2014). Similarly, in some HCMV-seropositive individuals carrying a heterozygous or homozygous deletion of KLRC2 there is also an expansion of the unique subset of NK cells lacking FcεRIγ (Liu et al., 2016; Muntasell et al., 2016). KLRC2−/− NK cells in HCMV-seropositive individuals display a mature phenotype (LILRB1highCD7lowCD161lowCD57highFcεR1γlowNKG2A−), which is similar but not identical to the CD94-NKG2Chigh NK cell subset in HCMV-seropositive KLRC2+/+ individuals (Liu et al., 2016). More importantly, these mature KLRC2−/− NK cells share enhanced TNF and IFN-γ production against antibody-coated target cells, impaired IFN-γ production after IL-12 plus IL-18 stimulation, and an epigenetic remodeling associated with demethylation of CpG motifs in the IFNG promoter region, similar to memory CD94-NKG2Chigh NK cells in HCMV-seropositive KLRC2+/+ individuals (Liu et al., 2016). The identification of an NK cell subset with memory features in HCMV-seropositive individuals lacking expression of NKG2C raises the possibility that inflammatory cytokines during HCMV infection may induce the differentiation of these mature NKG2C-deficient NK cells with augmented effector functions, which is reminiscent of the cytokine-activated Ly49H−KLRG1high NK cell subset in MCMV-infected mice.

Of note, both memory NK cells and cytokine-activated NK cells have several functional properties that may be beneficial for cancer immunotherapy, including augmented effector functions against tumors, persistence in vivo, and the capacity for secondary expansion. Further studies of the transcriptional signature and epigenetic modifications defining these distinct NK cell subsets are needed to better understand the critical regulatory factors for the divergence, differentiation, maintenance, and functional properties of these NK cells and to provide important insights into the development of effective NK cell–based vaccination strategies against infectious diseases and malignancies.

Generation of NKp46-CreERT2 Tg mice

The bacterial artificial chromosome (BAC) clone RP23-267N11 that encodes mouse chromosome 7 was modified to insert an internal ribosome entry site (IRES)-CreERT2 cassette (provided by M. Shlomchik, Yale University School of Medicine, New Haven, CT) into the 3′ untranslated region of exon 7 of the mouse Ncr1 gene by a BAC recombineering technology using reagents, vectors, and bacterial strains available from the National Cancer Institute (Bethesda, MD; https://ncifrederick.cancer.gov/research/brb/recombineeringInformation.aspx). The recombined BAC construct containing the IRES-CreERT2 element in the Ncr1 gene locus was microinjected into the pronucleus of single-cell fertilized zygotes of FVB/NJ mice. A founder mouse carrying the recombined BAC construct in its genome was intercrossed with Rosa26-YFP B6 mice or Rosa26-tdTomato B6 mice. The BAC transgenic mice (NKp46-CreERT2 Tg mice) carrying the Rosa26-YFP allele or the Rosa26-tdTomato allele, which express YFP or tdTomato, respectively, upon CreERT2-mediated excision of loxP-flanked stop codon were treated with tamoxifen (oral gavage of 200 µg/g body weight in corn oil) for 4 or 5 consecutive days.

MCMV and L. monocytogenes infection

A stock of Smith strain MCMV was prepared by homogenizing salivary glands harvested from infected BALB/c mice as described previously (Brune et al., 2001). Mice were infected by intraperitoneal injection of 5 × 103 PFU of salivary gland virus. In some experiments, Smith strain WT MCMV and Δm157 mutant MCMV (Bubić et al., 2004; provided by U. Koszinowski, Max von Pettenkofer-Institut, Munich, Germany) were prepared by infecting C57BL/6 3T3 cells in cell culture as described previously (Bubić et al., 2004). Mice were infected by intraperitoneal injection of 5 × 105 PFU WT MCMV or 2.5 × 105 PFU Δm157 MCMV. NKp46-CreERT2 Tg mice carrying the Rosa26-YFP allele were treated with tamoxifen for 4 or 5 d after MCMV infection on day 0. In some experiments, mice were inoculated intraperitoneally with 200 µg of a neutralizing mAb against mouse IL-12 p40 (clone C17.8) or an isotype-matched control rat IgG2a on the day before MCMV infection, and on days 3 and 6 p.i. L. monocytogenes 10403S strain was grown in brain–heart infusion broth to an OD600 of 0.2, and mice were infected intravenously with 5 × 104 CFU. Dose was determined by CFU assays for each infection.

Preparation of single-cell suspensions and NK cells

Leukocytes were isolated from spleen, bone marrow, blood, liver, and salivary glands. Livers were isolated after tissue perfusion with PBS and homogenized by using a Dounce homogenizer, and lymphocytes were prepared using centrifugation on a 40 and 60% Percoll gradient (GE Healthcare). Sublingual glands and submandibular glands were homogenized, and lymphocytes were prepared using centrifugation on a 40 and 70% Percoll gradient. NK cells were enriched by incubating splenocytes with purified rat mAbs against mouse CD4, CD5, CD8, CD19, Gr-1, and Ter119, followed by anti–rat IgG antibodies conjugated to magnetic beads (QIAGEN), as described previously (Nabekura et al., 2014).

Flow cytometry

Fc receptors (CD16 and CD32) were blocked with 2.4G2 mAb before surface or intracellular staining with the indicated fluorochrome-conjugated mAbs or isotype-matched control antibodies (BD, eBioscience, BioLegend, or TONBO Biosciences). In some experiments, cells were fixed with Cytofix (BD), followed by permeabilization with 0.1% Triton X-100 in PBS, and then stained with Alexa Flour 647–conjugated anti-Ki67 (BD). Samples were acquired on a LSRII or a FACSCalibur (BD) and data were analyzed with FlowJo software (FlowJo).

Ex vivo stimulation of NK cells

100,000 NK cells were incubated in 96-well tissue culture plates coated with anti-NK1.1 (clone PK136) or isotype-matched control mouse IgG2a as described previously (Nabekura et al., 2015); incubated with 2.5 ng/ml mouse IL-12 and 2.5 ng/ml mouse IL-18 (R&D Systems); or co-cultured with 105 RMA, RMA expressing m157, or RMA expressing Rae1γ for 5 h at 37°C in the presence of PE-conjugated anti-CD107a mAb and GolgiStop (BD), followed by staining for surface molecules and intracellular IFN-γ as previously described (Nabekura and Lanier, 2014).

In vivo cytotoxic assay

Enriched NK cells were stained with antibodies against TCRβ, B220, Ly49H, and KLRG1. NK cells in the spleens of naive uninfected NKp46-CreERT2 Tg x Rosa26-YFP mice were purified by sorting YFP+KLRG1+ cells gated on non–T cell and –B cell lymphocytes by using a FACSAria III (BD) on day 21–25 after the tamoxifen injection. Memory NK cells and cytokine-activated NK cells in the spleens of MCMV-infected NKp46-CreERT2 Tg x Rosa26-YFP mice were purified by sorting YFP+Ly49H+KLRG1high cells and YFP+Ly49H−KLRG1high cells gated on non–T cell and –B cell lymphocytes by using a FACSAria III on day 26 p.i. 15,000 NK cells were injected intraperitoneally into DAP10 and DAP12 double-deficient B6 mice. DAP10 and DAP12 double-deficient B6 mice were depleted of CD4+ and CD8+ T cells on the day before transfer of donor NK cells by intraperitoneal injection of 100 µg of anti-CD4 (clone GK1.5) and 100 µg of anti-CD8 (clone 2.43) mAbs. RMA transfectants expressing Rae1γ (RMA-Rae1γ) and RMA-S cells were labeled with CellTrace Violet, mixed with unlabeled RMA cells at a ratio of 1:1:1 (105 of each cell line; a total of 3 × 105 cells), and the mixed tumor cells were injected intraperitoneally into DAP10- and DAP12-deficient B6 mice on the day of donor NK cell transfer. An aliquot of the mixed tumor cells was cultured as a control. After 48 h, peritoneal cavity cells were collected and stained with anti-NK1.1, anti-Ly49A, and anti–H-2Kb mAbs. Tumor cells were gated on the basis of their characteristic forward and side light scatter properties, and then gated on NK1.1− and Ly49Ahigh cells. The different tumor cell types were discriminated by staining of H-2Kb and CellTrace Violet: RMA cells, H-2Kb+ CellTrace Violet−; RMA-Rae1γ cells, H-2Kb+ CellTrace Violet+; and RMA-S cells, H-2Kb− CellTrace Violet+. NKG2D-mediated cytotoxicity against RMA-Rae1γ was quantified as the proportion of viable recovered RMA-Rae1γ cells relative to that of RMA cells, and loss of RMA-S cells confirmed efficient NK cell cytolytic activity.

Statistical methods

The Student’s t test was used to compare results. P < 0.05 was considered statistically significant.

Acknowledgments

We thank the Lanier laboratory for comments and discussions. We are grateful to Helen Lu, Hong-Erh Liang, Richard M. Locksley, and Mark S. Anderson (University of California, San Francisco, San Francisco, CA) for helpful comments on BAC recombineering, and Mark J. Shlomchik (Yale University School of Medicine, New Haven, CT) for providing the IRES-CreERT2 cassette.

The work was supported by National Institutes of Health grant AI068129. L.L. Lanier is an American Cancer Society Professor. T. Nabekura is supported by the Friends of Leukemia Research Fund and the Nakajima Foundation.

The authors declare no competing financial interests.

Footnotes

Abbreviations used:

HCMV

human cytomegalovirus

ILC

innate lymphoid cell

IRES

internal ribosome entry site

ITAM

immunoreceptor tyrosine-based activation motif

MCMV

mouse cytomegalovirus

MFI

mean fluorescence intensity

p.i.

post infection

RSV

respiratory syncytial virus

Submitted: 18 May 2016

Revision received 5 August 2016

Accepted: 27 September 2016

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